Terpenoids from Myrrh and Their Cytotoxic Activity against HeLa Cells

The oleo–gum resin of Commiphora myrrha (Nees) Engl. has a long history of medicinal use, although many of its constituents are still unknown. In the present investigation, 34 secondary metabolites were isolated from myrrh resin using different chromatographic techniques (silica flash chromatography, CPC, and preparative HPLC) and their structures were elucidated with NMR spectroscopy, HRESIMS, CD spectroscopy, and ECD calculations. Among the isolated substances are seven sesquiterpenes (1–7), one disesquiterpene (8), and two triterpenes (23, 24), which were hitherto unknown, and numerous substances are described here for the first time for C. myrrha or the genus Commiphora. Furthermore, the effects of selected terpenes on cervix cancer cells (HeLa) were studied in an MTT-based in vitro assay. Three triterpenes were observed to be the most toxic with moderate IC50 values of 60.3 (29), 74.5 (33), and 78.9 µM (26). Due to the different activity of the structurally similar triterpenoids, the impact of different structural elements on the cytotoxic effect could be discussed and linked to the presence of a 1,2,3-trihydroxy substructure in the A ring. The influence on TNF-α dependent expression of the intercellular adhesion molecule 1 (ICAM-1) in human microvascular endothelial cells (HMEC-1) was also tested for 4–6, 9–11, 17, 18, 20, and 27 in vitro, but revealed less than 20% ICAM-1 reduction and, therefore, no significant anti-inflammatory activity.

Nonetheless, a signal between distant H 3 -14 and H 3 -2 indicates these groups are located on the same side of the ring. The missing signal between H 3 -14 and H-5 together with the large coupling constant between H-5 and H-6b (δ H 2.35; 3 J H-5, H-6b = 13.5 Hz) suggests opposite axial positions of H 3 -14 and H-5. Missing signals between axial H-5 to the isopropenyl substituent H 2 -12/H 3 -13 indicate that its location is on the other side of the cyclohexane (Figure 2d). Thus, the molecule is rel-8S-acetyloxy-7R-hydroxy-5R,10R-β-elemene. Related structures, such as β-elemene itself, are known as constituents of myrrh [42], whereas the substitution pattern of 3 is described here for the first time.
NOESY experiments were carried out to determine the relative configuration of the three stereocenters in compound 4. The substituents in pos. 2 and 4 must be on opposite sides of the ring due to the strong NOESY correlations between H 3 -1 to H-3b (δ H 2.28) and H-3b to H-4. The coupling constants of H-2/H-3 a,b and H-5b (δ H 2.72)/H-4 being less than 5 Hz indicate their equatorial position, resulting in an opposing orientation that necessitates their location under and above the ring, respectively. This corroborates the position of H 3 -13 on the same side as H 3 -15, due to the strong NOESY signal to H-5a (δ H 2.24), whereas H-11 correlates with H-5b ( Figure 3c). Thus, the configuration of compound 4 was determined to be rel-2R,4S,11R. 1), 134.8 (C-6), 123.3 (C-7), 152.6 (C-8), 140.1 (C-10)) and a carbonyl carbon (δC 178.6 (C 12)) ( Table 2). On the basis of 2D NMR spectra, the structure was identified as a cadinane type sesquiterpene with an aromatic B-ring, a methoxy function in pos. 2 and a γ-lacton moiety, previously described as commiterpene D [18] (Figure 3a). In accordance with no menclature established by Xu et al. [13], compound 4 was named commiterpene E. NOESY experiments were carried out to determine the relative configuration of th three stereocenters in compound 4. The substituents in pos. 2 and 4 must be on opposit sides of the ring due to the strong NOESY correlations between H3-1′ to H-3b (δH 2.28) and H-3b to H-4. The coupling constants of H-2/H-3a,b and H-5b (δH 2.72)/H-4 being less tha 5 Hz indicate their equatorial position, resulting in an opposing orientation that necessi tates their location under and above the ring, respectively. This corroborates the positio Compounds 5 and 6 (2.9 and 6.5 mg) were isolated as white crystals. The positivemode HRESIMS suggested that they were isomers with the same molecular formula of C 16  ; δ C 132.1 (C-1), 134.8 (C-4), 124.9 (C-5), 162.0 (C-7), 135.4 (C-10), 125.9 (C-11)), and one carbonyl carbon (δ C 173.5 (C-12)) (  ) and one carbonyl carbon (δ C 173.6 (C-12)) ( Table 2), indicating that 5 and 6 are diastereomers.
Compound 8 (0.9 mg) was obtained as a white solid, and its molecular formula was determined with positive-mode HRESIMS to be C 32 H 40 O 6 (m/z 521.2904 [M+H] + , calc. 521.2898). Based on NMR spectra, a dimerized sesquiterpenoid was identified and the following signals were assigned: two methoxy groups (δ and ten quaternary carbons, of which two were aliphatic including the spiro carbon (δ C 36.1 (C-10), 42.0 (C-10 )), six were aromatic (δ C 122.5 (C-7), 159.0 (C-8), 121.1 (C-11), 117.6 (C-7 ), 153.5 (C-8 ), 128.6 (C-11 )) and two were carbonyl carbons (δ C 196.5 (C-6), 202.4 (C-6 )) ( Table 3). 023, 28, x FOR PEER REVIEW derivatives 10 and 11, whose relative configurations have already been deter Greve et al. [15]. This indicates that the two pairs of diastereomers correspon configurations. Furthermore, the strong similarity of 5 to 10 and 6 to 11 can be c by their CD spectra (Figure 4). For a safe stereochemical determination of both s ters per molecule, the CD spectra of both possible diastereomeres would be n Although, as the spectra are both so closely corresponding to one diastereomer substituent-differing compounds, the authors are daring to propose the same rel figurations: a (rel-2R,8S) configuration can be postulated for 5, whereas for com a (rel-2R,8R) configuration can be assumed. In analogy to 10 and 11, the semitriv 2β-methoxyglechomanolide and 8-epi-2β-methoxyglechomanolide are suggeste previously unknown compounds 5 and 6.    Compound 8 (0.9 mg) was obtained as a white solid, and its molecular determined with positive-mode HRESIMS to be C32H40O6 (m/z 521.2904 521.2898). Based on NMR spectra, a dimerized sesquiterpenoid was ident following signals were assigned: two methoxy groups (δH 3. 33   Analysis of 2D NMR data revealed a dimer composed of a guaiane and a germacrenetype sesquiterpene, connected by a four-membered spirocycle ( Figure 6a). More precisely, the spectra revealed a structure resembling commiphorine A [21]. The guaiane moiety (part I) is the C-1 epimer of the known monomer guaiane myrrhterpenoid O (22) [15] and is, thus, the same as described for the dimers commiphorine A and commiphoratone B [22]. Part (II) varies from commiphorine A. First, the double bond between C-1 and C-2 is E-configurated, deduced from NOESY signals as well as the large coupling constant of 3 J H-1', H-2' = 16.0 Hz. Second, an additional methoxy substituent is located at C-3 . Third, according to the NOESY signals, the stereochemistry at the spiro-C-10 is reversed, resulting in contrary axial chirality. The germacrene moiety (part II) was also isolated as a monomer (14), which is already known for C. myrrha as 3S-methoxy-4R-furanogermacra-1E,10(14)dien-6-one [43,44]. Key HMBC signals between H-9 to C-1 /C-10 and H-9b' to C-9 indicate a connection between the two sesquiterpene monomers (Figure 6a).
is E-configurated, deduced from NOESY signals as well as the large coupling constant of 3 JH-1', H-2' = 16.0 Hz. Second, an additional methoxy substituent is located at C-3′. Third, according to the NOESY signals, the stereochemistry at the spiro-C-10′ is reversed, resulting in contrary axial chirality. The germacrene moiety (part II) was also isolated as a monomer (14), which is already known for C. myrrha as 3S-methoxy-4R-furanogermacra-1E,10(14)-dien-6-one [43,44]. Key HMBC signals between H-9 to C-1′/C-10′ and H-9b' to C-9 indicate a connection between the two sesquiterpene monomers (Figure 6a). The relative configuration of compound 8 was partially elucidated by the NOESY experiments. Its spatial arrangement at C-10' is deviating from that of commiphorine A indicated by signals between H-9 and H-1′/H-2′ on the one hand and the correlation between H-1 and H-9a' on the other hand ( Figure 6b). Due to the distance, the configuration of the stereocenters C-3′ and C-4′ was not determinable in relation to the rest of the molecule, resulting in four possible absolute configurations. Therefore, the CD spectrum was compared to those of related molecules, and the determination of the absolute configuration was then attempted via EDC calculations.
As described above, compound 8 was found to be closely related to commiphorine A, reported by Dong et al. in 2019 [21] as a constituent of myrrh resin (Resina Commiphora). The authors also published the absolute configuration of their compound, which was based on the comparison of the measured electronic CD (ECD) spectrum with simulated quantum-mechanical computations (see Figure 7). The relative configuration of compound 8 was partially elucidated by the NOESY experiments. Its spatial arrangement at C-10' is deviating from that of commiphorine A indicated by signals between H-9 and H-1 /H-2 on the one hand and the correlation between H-1 and H-9a' on the other hand ( Figure 6b). Due to the distance, the configuration of the stereocenters C-3 and C-4 was not determinable in relation to the rest of the molecule, resulting in four possible absolute configurations. Therefore, the CD spectrum was compared to those of related molecules, and the determination of the absolute configuration was then attempted via EDC calculations.
As described above, compound 8 was found to be closely related to commiphorine A, reported by Dong et al. in 2019 [21] as a constituent of myrrh resin (Resina Commiphora). The authors also published the absolute configuration of their compound, which was based on the comparison of the measured electronic CD (ECD) spectrum with simulated quantum-mechanical computations (see Figure 7).
The ECD spectrum simulated in the present study for the postulated structure of commiphorine A [21] (Figure 7d, blue curve) is opposite to the experimental spectrum and simulation of the previous authors (Figure 7c, blue curve); i.e., the Cotton effect (CE) at the longest wavelength (about 290 nm) is positive, not negative. Due to this discrepancy, the structural models underlying the published ECD, shown in the Supplementary Materials of [21], were carefully inspected. There, the models used a compound with a 1,5-cisguaianolide moiety, as opposed to the 1,5-trans structure postulated for commiphorine A, as determined from NMR spectroscopy (see Figure S1, Supplementary Materials). For comparison, we also simulated the ECD spectrum using the 1,5-cis-configured structures (see Figure S1, Supplementary Materials). These molecular models (from the original coordinates published in the Supplementary Materials of [21]) yield an ECD spectrum with a negative CE at their lowest energy transition, but they do not reflect the postulated 1,5-trans-configured chemical structure.
In conclusion, the experimental ECD spectrum of commiphorine A actually corresponds to the enantiomeric structure postulated in [21]. The ECD spectrum of the enantiomer (red line in Figure 7d) fits the compound's experimental spectrum (red line in Figure 7c). On these grounds, we revised the structure of commiphorine A to be the enantiomer of the one previously published (red structure in Figure 7a).

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10 of 31 Figure 7. (a) Blue: Structure of commiphorine A according to [21], red: Enantiomeric structure; (b) minimum energy conformer of the blue structure depicted in a after a conformational search and full energy minimization (MMFF94x force field/low mode dynamics/energy minimization by MO-PAC/PM3 followed by DFT B3LYP/6−31G(d,p)). (c) Experimental ECD spectrum of commiphorine A (red curve) and simulated ECD spectrum (blue curve) according to [21]. (d) Blue: ECD spectrum simulated by TDDFT (B3LYP/6−31G(d,p)) in the present study with the postulated [21] structure of commiphorine A (blue structure in a). Red: Simulated ECD spectrum of the enantiomer of the postulated [21] structure (red structure in a). Note the very close fit of the red curve with the experimental spectrum shown in c.
The other compounds were also elucidated from their NMR spectra and ide described in the literature: glechomanolide (9) [48], 2α-methoxy-6-oxog Compounds 10 and 11 (10.9 and 11.3 mg) were identified as 2β-acetyloxyglechomanolide and 8-epi-2β-acetyloxyglechomanolide by matching their NMR data with the ones published by Greve et al. who isolated them in a 2:1 mixture from C. myrrha [15]. In the present work, these compounds were separated for the first time with preparative HPLC on a biphenyl column and could be characterized by UV, CD spectra and polarimetry.

Triterpenoids
The structures of all isolated triterpenoids are presented in Figure 9. and myrrhterpenoid O (22). For myrrhterpenoid O, NOESY data were too weak for a firmation of the configuration. This molecule is probably the correct structure of 2-m oxyfuranoguaia-9-ene-8-one, first published in 1983, as their NMR signals are cons (Table S5) [15,45].
The NMR data of compound 23 show many similarities to another isolated triterpene (33) with one major deviation; H-3 is shifted downfield to 4.77 ppm versus 3.32 ppm in 33. This effect was also observed in the other O-substituted triterpenes 30-32, which also show downfield protons at the substituted hydroxyl groups. The molecular formulas determined by HRESIMS and the additional NMR signals of 30-32 confirm a substitution with an isovalerate moiety. Additionally, the position of the substituent is verified by a HMBC signal between H-3 and C-1 (see also Tables S7 and S8). The relative configuration of compound 23 in the A ring was determined based on coupling constants and NOESY correlations. A relatively high coupling constant of 9.9 Hz between H-3, -2 and -4 indicates an axial position of these three protons, while the lower coupling constant between H-1 and -2 ( 3 J H-1, H-2 = 2.9 Hz) infers an axial-equatorial coupling and, thus, an equatorial position of H-1. This can be confirmed by NOESY signals between H-1 and -2 as well as -19 and between H-3 and -29 ( Figure 10).
The relative configuration of compound 23 in the A ring was determined based on coupling constants and NOESY correlations. A relatively high coupling constant of 9.9 Hz between H-3, -2 and -4 indicates an axial position of these three protons, while the lower coupling constant between H-1 and -2 ( 3 JH-1, H-2 = 2.9 Hz) infers an axial-equatorial coupling and, thus, an equatorial position of H-1. This can be confirmed by NOESY signals between H-1 and -2 as well as -19 and between H-3 and -29 ( Figure 10). Compound 23 can, thus, be assigned a rel-(1α,2α,3β) configuration and, reflecting trivial names of known triterpenes, the name 3β-isovaleroyloxy-29-nor-lanost-8,24-diene 1α,2α-diol is suggested for the hitherto-unknown substance.  (Table 4) The NMR data of compound 24 show many similarities to 33, such as the doublet for H-29 (0.97 ppm) and double bonds between C-8, -9, -24 and -25, indicating a 29-nor-lanost 8,24-diene backbone. In comparison with 33, two protons and carbons in pos. 1 and 2 are shifted in high field (δH 3.22 and 3.12 ppm (24) compared to 3.94 and 3.62 ppm (33); and δC 59.6 and 57.5 ppm (24) versus 74.9 and 73.8 ppm (33)). These chemical shifts are typica for an epoxide and indicate an epoxidation of compound 24 at C-1/-2, which is confirmed by the HRESIMS formula.
Stereochemistry in the A ring was deduced from NOESY signals showing correlations between H3-18 and H-19 to confirm a β-configuration of the methyl group at pos. 19 Analogously, a correlation between H-5 and H3-28 indicates an α-configuration of H-5 Thus, the orientation at pos. 3 and 4 can be clarified by signals above (H3-19, H-4) and below (H-3, -5) the ring (Figure 11).    (Table 4) The NMR data of compound 24 show many similarities to 33, such as the doublet for H-29 (0.97 ppm) and double bonds between C-8, -9, -24 and -25, indicating a 29-nor-lanost-8,24-diene backbone. In comparison with 33, two protons and carbons in pos. 1 and 2 are shifted in high field (δ H 3.22 and 3.12 ppm (24) compared to 3.94 and 3.62 ppm (33); and δ C 59.6 and 57.5 ppm (24) versus 74.9 and 73.8 ppm (33)). These chemical shifts are typical for an epoxide and indicate an epoxidation of compound 24 at C-1/-2, which is confirmed by the HRESIMS formula.
Stereochemistry in the A ring was deduced from NOESY signals showing correlations between H 3 -18 and H-19 to confirm a β-configuration of the methyl group at pos. 19. Analogously, a correlation between H-5 and H 3 -28 indicates an α-configuration of H-5. Thus, the orientation at pos. 3 and 4 can be clarified by signals above (H 3 -19, H-4) and below (H-3, -5) the ring ( Figure 11).
Molecules 2023, 28, x FOR PEER REVIEW 15 of Figure 11. Key NOESY signals of 24 for determination of the relative configuration in the A ring.
Furthermore, the orientation of the epoxy moiety is of interest, because a cis or tra configuration is possible. The cis arrangement is characterized by an equatorial positi of the protons H-1 and -2, while in the trans configuration, they are axially apart. H-1 an H-2 show a strong NOESY signal, which is atypical for axially located protons. Furthe more, correlations of both protons to groups above (H3-19) or below (H-3) the ring can observed (Figure 12) also suggesting an equatorial orientation. Consequently, a cis confi uration of the epoxy moiety is postulated. Our methods cannot determine whether t epoxide is in the α or β position, as this does not affect the orientation of the molecule Furthermore, the orientation of the epoxy moiety is of interest, because a cis or trans configuration is possible. The cis arrangement is characterized by an equatorial position of the protons H-1 and -2, while in the trans configuration, they are axially apart. H-1 and H-2 show a strong NOESY signal, which is atypical for axially located protons. Furthermore, correlations of both protons to groups above (H 3 -19) or below (H-3) the ring can be observed ( Figure 12) also suggesting an equatorial orientation. Consequently, a cis configuration of the epoxy moiety is postulated. Our methods cannot determine whether the epoxide is in the α or β position, as this does not affect the orientation of the molecule in space, and thus the NMR data ( Figure 12). Compound 24 has not been described in the literature, and, therefore, the name 29-nor-1,2-cis-epoxy-lanost-8,24-diene-3β-triol is suggested.
Furthermore, the orientation of the epoxy moiety is of interest, because a cis or tra configuration is possible. The cis arrangement is characterized by an equatorial positio of the protons H-1 and -2, while in the trans configuration, they are axially apart. H-1 an H-2 show a strong NOESY signal, which is atypical for axially located protons. Furthe more, correlations of both protons to groups above (H3- 19) or below (H-3) the ring can observed (Figure 12) also suggesting an equatorial orientation. Consequently, a cis confi uration of the epoxy moiety is postulated. Our methods cannot determine whether th epoxide is in the α or β position, as this does not affect the orientation of the molecule space, and thus the NMR data ( Figure 12). Compound 24 has not been described in th literature, and, therefore, the name 29-nor-1,2-cis-epoxy-lanost-8,24-diene-3β-triol is su gested. Compound 30 (2.0 mg) was obtained as white crystals and assigned a molecular fo mula of C32H52O3 by HRLIFDIMS (m/z 484.3900 M + , calc. for 484.3911). The NMR data el cidated that the structure and relative configuration of 30 are in accordance with a com pound isolated in 1988 by Provan et al. [58]. The complete data set of α-acetoxy-9,19-c clolanost-24-ene-3β-ol (30) is presented here for the first time (Table S7, Figure S3).

The Cytotoxicity of Selected Compounds against HeLa Cells
For investigation of their biological activity, selected and mainly sesquiterpenoid compounds, which were obtained in sufficient amount and HPLC-DAD purity (>90%) (4-6, 9-11, 17, 18, 20 and 27), were tested in an ICAM-1 in vitro assay, as described before [66]. In this assay, only a small reduction (less than 20%) of TNF-α dependent ICAM-1 expression in HMEC-1 cells was observed, and, hence, no relevant anti-inflammatory activity could be detected. The results are provided in the Supplementary Material in Figure S14.
Additionally, cytotoxic effects of selected isolates (2, 26-29, 31, 33, and 34) on human cervical cancer cells (HeLa) were studied with special attention to the triterpenoids. For this, an MTT viability assay was used and carried out with some modifications according to Mosman [67] monitoring the viability of the cells via their (mitochondrial) reductase activity. The results are presented in relation to the untreated control (u.c.). To exclude the possibility of solvent effects, cells were also treated with the highest used DMSO concentration (negative control).
to Mosman [67] monitoring the viability of the cells via their (mitochondrial) reductase activity. The results are presented in relation to the untreated control (u.c.). To exclude the possibility of solvent effects, cells were also treated with the highest used DMSO concentration (negative control).

Discussion and Conclusions
The presented 34 terpenoids from myrrh are only a small excerpt of its secondary metabolites and illustrate their broad heterogeneity. Sesquiterpenes of six different structural types (germacranes, eudesmanes, seco-eudesmanes, elemanes, cadinane, and guaiane) were found, in addition to one sesquiterpene dimer and triterpenoids of four different scaffolds (mansumbinanes, lanostanes, dammaranes, and cycloartanes). The analysis of the structural richness is crucial for the further development of the standardized quality analytics of the drug, which are still based on a TLC experiment detecting the volatile sesquiterpenes in the European Pharmacopoeia [68]. The examination of the composition is also the basis for finding the molecular targets of the traditionally used drug, which are still unknown.
Concerning the sesquiterpene dimer (8) analytics of the drug, which are still based on a TLC experiment detecting the volatile sesquiterpenes in the European Pharmacopoeia [68]. The examination of the composition is also the basis for finding the molecular targets of the traditionally used drug, which are still unknown.
Concerning the sesquiterpene dimer (8), the accompanying isolation of its monomers with a double bond at the linking position strongly supports Dong et al.'s (2019) suggestion of a 2,2-cycloaddition as biosynthetic pathway (Figure 14) [21]. To test the anti-inflammatory activity of the isolated molecules, the ICAM-1 expression on human endothelial cells was chosen as a pathway. Interestingly, one of the active compounds found in earlier work [18] only differs in a missing double bond from as inactive analysed compounds 18 (position C- 8,9) or an additional one from 20 (position C-1,2). The negative results of all the tested substances indicate that the proven anti-inflammatory activity of myrrh is either caused by other compounds [18] or triggered via another pathway. Investigation of the molecular mechanisms of action, therefore, remains an interesting field for further research.
The observed cytotoxic effect of compounds 26, 29 and 33 on HeLa cells provide a first hint of cytotoxic activity of these myrrh triterpenoids against cancer cells. For a more comprehensive and robust investigation of cytotoxicity a broader set of triterpenoids as well as a second quantitative analytical method for purity control, must be included. Interestingly, an A ring cleavage of mansumbinol (26) resulting in a carboxylic acid and an isopropenyl partial structure led to a complete loss of activity for compound 27. Compound 29, with three hydroxyl groups at C-1-3, showed significant cytotoxicity, whereas similar substances with a different substitution pattern in the A ring, as in 28 or 31, were less potent. This was also observed for 33, which is a lanostane-type triterpene but corresponds to 29 with the A ring substitution. Thus, it can be hypothesized that the 1,2,3-trihydroxy substructure in the A ring contributes to a cytotoxic effect, whereas the type of triterpene skeleton has less influence on the activity. To test the anti-inflammatory activity of the isolated molecules, the ICAM-1 expression on human endothelial cells was chosen as a pathway. Interestingly, one of the active compounds found in earlier work [18] only differs in a missing double bond from as inactive analysed compounds 18 (position C- 8,9) or an additional one from 20 (position C-1,2). The negative results of all the tested substances indicate that the proven antiinflammatory activity of myrrh is either caused by other compounds [18] or triggered via another pathway. Investigation of the molecular mechanisms of action, therefore, remains an interesting field for further research.
The observed cytotoxic effect of compounds 26, 29 and 33 on HeLa cells provide a first hint of cytotoxic activity of these myrrh triterpenoids against cancer cells. For a more comprehensive and robust investigation of cytotoxicity a broader set of triterpenoids as well as a second quantitative analytical method for purity control, must be included. Interestingly, an A ring cleavage of mansumbinol (26) resulting in a carboxylic acid and an isopropenyl partial structure led to a complete loss of activity for compound 27. Compound 29, with three hydroxyl groups at C-1-3, showed significant cytotoxicity, whereas similar substances with a different substitution pattern in the A ring, as in 28 or 31, were less potent. This was also observed for 33, which is a lanostane-type triterpene but corresponds to 29 with the A ring substitution. Thus, it can be hypothesized that the 1,2,3-trihydroxy substructure in the A ring contributes to a cytotoxic effect, whereas the type of triterpene skeleton has less influence on the activity.
To our knowledge, this is the first time that myrrh triterpenes are reported to have cytotoxic effects against HeLa cells. Previous investigations occasionally describe the cytotoxic activity of myrrh oil or single myrrh sesquiterpenes on both normal and cancer cells, but activity is often selective for the former [33,69]. Further cell viability experiments with other tumour and noncancer cell lines will be necessary to characterize their activity better, especially regarding possibly selective anticancer activity or general cytotoxicity.

Isolation
The isolation process was carried out mostly as published [18] and is, thus, roughly summarized and complemented with the new procedures.

Liquid-Liquid Partition
Portions of ethanolic extract were solved in methanol and partitioned with n-heptane in a separatory funnel to gain a methanol (MeOH, 328.13 g) and an n-heptane-soluble fraction (HEP, 69.99 g).

Solid Phase Extraction: MeOH Fraction
To further divide the methanol fraction, solid-phase extraction by silica gel (Geduran Si 60 (0.063-0.200 mm), Merck Chemicals, Darmstadt, Germany) was performed. Therefore, 150 g of MeOH fraction was mortared with silica gel and submitted dryly in portions on wet-packed columns. The first elution step with ethyl acetate resulted in fraction M1 (107.0 g) and the second step with methanol in M2 (42.3 g).

Centrifugal Partition Chromatography (CPC)
A Spot centrifugal partition chromatography (CPC) device with a 250 mL rotor (Armen Instrument, Paris, France), a 510 HPLC pump (Waters GmbH, Eschborn, Germany) and a 2111 Multirac Fraction Collector (LKB-Produkter AB, Bromma, Sweden) was used for the separation of fractions F5-7, F9 and M1.2. The separation was performed at a rotation speed of 1000 rpm and a flow rate of 5 mL/min, with a solvent system consisting of n-hexane, acetonitrile and methanol (40/25/10 v/v/v) [70], and was executed in two stages. First, the lower phase (LP) was used as a stationary phase in ascending mode (ASC) for 800 mL for F5-7/F9 or 925 mL for M1.2, respectively. Second, phases were switched, and the process was conducted in descending mode (DSC) for another 200 mL or 275 mL, respectively. Subsequently, subfractions (F5C1-6, F6C1-8, F7C1-8, F9C1-4, and M1.2C1-7) were formed, of which the following were processed further: fraction (mode, retention volume, and weight):

Preparative HPLC
For the isolation of pure compounds, a preparative high-performance liquid chromatography (HPLC) device equipped with a 1260 Infinity binary pump, a 1260 Infinity manual injector, a 1260 Infinity fraction collector, a 1260 Infinity diode array detector (all Agilent Technologies, Santa Clara, USA) and a Kinetex ® column (Biphenyl, 100 Å, 5 µm, 21.2 × 250 mm, Phenomenex, Aschaffenburg, Germany) at a flow rate of 21 mL/min or-for fractions M1.2C6 and M1.2C7-a Nucleodur TM C18 Isis column (RP18, 5 µm, 10 × 250 mm, Macherey-Nagel, Düren, Germany) at a flow rate of 5 mL/min was used. Separation was achieved by gradients consisting of acetonitrile (A)/water (B) and peaks were detected at 200 nm. After the elimination of acetonitrile via evaporation, the water fractions were partitioned four times with diethyl ether or ethyl acetate (M1.2C6 and M1.2C7) and organic phases were dried under a nitrogen stream. The gradients used for the separation can be found in Table 5.

Optical Methods
For further characterization of the isolates, optical data were gathered by using solutions in methanol. Specific optical rotation was measured by an UniPol L 1000 polarimeter (Schmidt + Haensch GmbH & Co., Berlin, Germany) using a microtube (50 mm, 550 µL) at 589 nm. UV-spectra were recorded by a Cary 50 Scan UV-spectrophotometer (Varian Deutschland GmbH, Darmstadt, Germany) in a quartz cuvette (QS, 1.0 cm, Hellma GmbH & Co. KG, Müllheim, Germany) in a range of 200-800 nm. Additionally, CD spectra were measured on a J-715 spectropolarimeter (JASCO Deutschland GmbH, Gross-Umstadt, Germany) with a 0.1 cm quartz cuvette (Type: 100-QSQ, Hellma GmbH & Co. KG). Thereby, ten scans were recorded at 22 • C from 190 to 300 or 400 nm with a scanning rate of 50, 100 or 200 nm/min in 0.5 nm steps and the Savitzky-Golay algorithm was used for spectra smoothing (convolution width: 15).

Simulation of ECD Spectra
Molecular models of commiphorine A and compound 8 (commiphorine C) were generated with the Molecular Operations Environment (MOE, v. 2020.09, Chemical Computing Group, Montreal, Canada) using the MMFF94x force field. After a conformational search with the low mode dynamics (LMD) method, the lowest energy conformers were energy-minimized with the semiempirical method PM3. In each case, the lowest energy conformer was used for the spectra simulation. The structure was exported to Gaussian (v. Gaussian03W, Gaussian Inc., Pittsburgh, Pa., U.S.A.) and fully minimized by density functional theory (DFT) with the B3LYP density functional and the 6−31G(d,p) basis set. The minimized structures were then subjected to a time-dependent DFT (TDDFT) calculation using the same functional and basis set, which yielded the electronic transition vectors determining the ECD spectra. Typically, the first (i.e., lowest in frequency) 12-18 electronic transitions were computed. The Gaussian output for electronic transition energies (E in eV) and rotator strengths (R, dipole length in cgs) was used to simulate ECD spectra by applying a Gaussian shape function with a band width at 1/e height σ = 0.15 eV. The resulting spectra shown in Figures 8 and 9 were scaled by factor 0.5 and blue-shifted by 0.15 eV.
The structures of three conformers of 1,5-cis-configured "commiphorine A" were built directly from the atomic coordinates published by the authors of the previous study [21] in their supplementary file. The ECD spectra of these models were simulated as described above. There was no significant difference between the three spectra, and so only the spectrum of structure 1a1 [21] is shown in Figure S1.

Purity
The purity of isolates was determined through HPLC-DAD analysis (190-400 nm for the HEP fraction or 200-400 nm for the MeOH fraction) on an Elite LaChrom system consisting of an autosampler L-2200, a pump L-2130, a column oven L-2350, a diode array detector L-2455 (all Hitachi, Tokyo, Japan) and a Kinetex ® biphenyl column (100 Å, 5 µm, 4.6 × 250 mm, Phenomenex, Aschaffenburg, Germany) or a Nucleodur TM C18 Isis column (RP18, 5 µm, 4.6 × 250 mm, Macherey-Nagel, Düren, Germany). For analyses, gradients described in 0 were conducted at a flow rate of 1 mL/min and an injection volume of 5 or 10 µL (acetonitrile, 1 mg/mL). Thus, the purity was calculated as the proportion of the integral of the main peak in the chromatogram using the maxplot adjusted by a blank (EZChrom Elite 3.1.7, Hitachi).
The human microvascular endothelial cell line (HMEC-1) was provided by Dr. E. Ades und F.-J. Candel (CDC, Atlanta, GA, USA), as well as Dr. T. Lawley (Emory University, Atlanta, GA, USA). These cells were cultured in EASY Endothelial Cell Growth Medium supplemented with 10% FBS, 50 ng/mL amphotericin B and 50 ng/mL gentamicin.
For both the used cell lines, mycoplasma contamination was excluded by PCR and culture from GATC Biotech AG (Konstanz, Germany) and they were cultured in an atmosphere of 5% CO 2 and 90% relative humidity at 37 • C.

MTT Assay
The MTT assay was conducted with both cell lines and differed (besides the different respective medium) only in the seeded cell density: HeLa experiments, 8 × 10 4 cells/well, HMEC-1 experiments 9 × 10 4 cells/well. They were seeded (in 100 µL/well) in 96-well plates and incubated at 37 • C in an atmosphere of 5% CO 2 and 90% relative humidity. After 24 h, the medium was replaced by a sample solution in medium (25-100 µM, max. 0.2% DMSO, v/v) and kept for another 24 h at the same conditions. Subsequently, the supernatant was removed and 100 µL of an MTT solution in medium (0.4 mg/mL) was added and incubated for three hours. Thereafter, cells were treated with 10% sodium dodecyl sulfate (SDS) in water and stored at room temperature in the dark until the formazan crystals were dissolved and the absorbance at 560 nm could be determined using a Tecan microplate reader (Tecan Trading AG, Maennedorf, Switzerland). The cell viability was calculated as proportion compared to the average absorbance of the negative control group (only medium). To preclude the possibility of solvent effects, some cells were also treated with the highest used DMSO concentration. All tests were performed three times independently in hexaplicates.

ICAM-1 Assay
This assay was performed as previously described [66]. Confluently grown HMEC-1 cells from a culture flask (150 cm 2 ) were split (1:3), suspended in 13 mL of medium, and seeded in a 24-well plate (500 µL/well). After cultivation for 48 h at 37 • C in an atmosphere of 5% CO 2 and 90% relative humidity and the formation of a monolayer, the supernatant was replaced by substance dilutions in medium (6-70 µM) containing a maximum of 0.15% DMSO (v/v) and incubated for 30 min before stimulation with TNFα (10 ng/mL). In each performance, an unstimulated negative control (0.15% DMSO, v/v), an untreated control (medium), and a positive control (parthenolide, 5 µM) were included. 24 h later, the cells were washed with phosphate-buffered saline (PBS), detached by trypsin/EDTA, fixed by formalin 10% for 15 min, and treated with a murine fluorescein-isothiocyanate (FITC)-marked IgG1 antibody against ICAM-1 (Bio-Rad, Kidlington, UK) for 30 min. The cell suspension in PBS was analysed by a FACSCanto II (BD, Lakes, NJ, USA) (Flow 60 µL/min, FSC: 1 V, SSC: 320 V; FITC: 320 V). The ICAM-1 expression was calculated as a proportion of the mean obtained for the untreated control. The test was performed three times independently in duplicate.

Statistics
Significance levels were calculated in a one-way ANOVA followed by Tukey-HSD in SPSS 26 (IBM, Armonk, NY, USA), whereas nonlinear regression curves were determined by GraphPad Prism 5.0.0 (GraphPad Software, San Diego, CA, USA).